The demise of the crossing, which has been a freight-only service for the past eight years, was hastened by a fire which broke out onboard the Finlandia Seaways freight ship last week.
The blaze, which left one crew member injured, put the vessel out of operation for several months.
In the wake of the incident, DFDS Seaways, one of Europe’s largest shipping and logistics firms, said it had decided to end the service altogether.
In what represents a blow for Scottish trade at a time when uncertainty surrounding Brexit remains rife, the firm’s senior president, Kell Robdrup, said it had been left with no alternative.
“We are extremely sorry for the effect this has on our customers and partners in Scotland and Belgium,” he said.
“In cooperation with the Scottish Government and the port, we have tried everything in our power to save the route.
“This included going from a combined passenger and cargo ship to a freight ship, reducing costs by enabling double stacking of containers and reducing fuel costs by installing a scrubber to remove sulphur from the exhaust gas instead of using expensive, low-sulphur fuel.
“However, the route continued to make losses. And with the new situation with the ship out of service for months, the market, the customers and the financial situation will be negatively affected, and make a turnaround and a reopening unrealistic.”
The collapse of the route brings to an end its 16 year long history. It began operating as a combined freight and passenger ferry service in 2002, only to be reduced to a freight-only crossing in 2010 after DFDS cited insufficient passenger demand.
The timetable for the service’s full closure was quickened by last Monday’s fire in the engine room of the Finlandia Seaways ro-ro ship.
The injured crew member was airlifted to hospital where he is being treated for injuries caused by smoke inhalation.
A subsequent inspection identified “substantial damage” to the vessel, with Mr Robdrup stating it would be out of service for “several months” for repair work.
He added: “Our search for a replacement vessel was fruitless and revealed that there are no suitable ships available.
“Unfortunately, this will bring about further losses on the route, and it means that we have lost all hope of being able to turn around the route’s loss-making situation.
Charles Hammond, group chief executive of Forth Ports, which owns and operates the port at Rosyth, said: “We are of course very disappointed that DFDS has closed the Rosyth to Zeebrugge freight ferry service.
“The service has a long history at Rosyth, operating since 2002, but has faced a number of challenges over the years.”
He stressed Scotland remained “well connected” with continental Europe via Grangemouth, the nation’s largest container terminal, and said it would “work hard” to ensure as many customers as possible could be accommodated via Grangemouth.